Mutational analysis of tyrosine-191 in the catalysis of Cephalosporium acremonium isopenicillin N synthase

Isopenicillin N synthase (IPNS) is a key enzyme responsible for the catalyt ic conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N in the p-lactam antibiotic biosynthetic pathway. The Asper gillus nidulans IPNS crystal structure implicated amino acid residues tyros ine-189, arginine-279, and serine-281 in the substrate-binding of the valin e carboxylate portion of ACV via hydrogen bonds. In previous reports, we pr ovided mutational evidence for the critical involvement of the correspondin g arginine-281 and serine-283, which constitute a conserved R-X-S motif, fo r the catalysis of Cephalosporium acremonium IPNS (cIPNS), In this study, w e report the site-directed mutagenesis of the corresponding tyrosine-191 in cIPNS to four amino acids from different amino acid groups, namely, phenyl alanine, serine, histidine, and aspartate. The mutants Y191F, Y191H, and Y1 91R respectively yielded specific activities at levels of 3, 8.6, and 18.8% relative to the wild-type when enzyme bioassays were performed using purif ied protein fractions. These results were surprising, as previous mutationa l analyses involving arginine-281 and serine-283 resulted in non-measurable specific activities, thus suggesting that tyrosine-191 is important but no t critical for the activity of cIPNS due to its involvement in ACV binding. Hence, it is likely that tyrosine-191 is the least critical of the three r esidues involved in binding the ACV valine carboxylate moiety.